1. Field of the Invention
The present invention relates to liquid chromatography and more specifically to a method of preventing contamination of a chromatography column from particulate and dissolved contaminants.
2. Background of the Invention
Liquid chromatography is a technique which employs a stationary phase and a liquid mobile phase to separate mixtures into various components of the mixture. A separation column generally consists of small beads or particles of stationary phase that have been slurried with a solvent and then packed into column hardware to form a chromatographic bed. The separating power of a liquid chromatographic column depends upon maintaining a uniform bed of packing material free of particulate and dissolved contaminants.
The use of column protection is required, especially when analyzing dirty or contaminated samples or samples containing components which irreversibly bind to the stationary phase. Continued injections of samples containing particulate or strongly retained, non-eluting solutes will eventually degrade the stationary phase and chromatographic performance of the separating column. Degradation in column performance is evidenced by changes in peak retention times, column efficiency and column selectivity.
Conventionally, two methods have been used to protect liquid chromatographic separation columns from contamination. The first method is use of a porous frit or series of frits to capture particulate material. Frits are generally made from porous stainless steel or glass, although other materials such as porous titanium and polymers such as polyether ether ketone (PEEK) or polypropylene may be used. A frit is normally located at each end of a separation column in order to contain the particulate packing material within the column. The frit at the head of a column also serves the function of trapping particulate material. A frit normally has pores ranging in size from 0.2 to 5 .mu.m. In addition to frits located at either end of a chromatographic column, a chromatographic system may also contain in-line frits located at various positions in the flow path. An in-line frit located between the sample injection valve and the separation column will also trap particulate material. A frit is replaced when it becomes plugged by particulate material and eluate back pressure increases to an unacceptable level.
Although useful, frits have a serious drawback. Frits have a low surface area and are inert and cannot trap or retain dissolved contaminants. Dissolved contaminants pass through the frit and are adsorbed or retained on the chromatographic stationary phase of the separation column. Continued contamination of the stationary phase degrades performance of the column.
The second method for protecting liquid separation columns is the use of guard columns or guard cartridges. A guard column or cartridge is connected in series upstream from a liquid chromatographic column to protect the separation column. The guard column is usually shorter than the separation column, but contains packing material which is similar to the stationary phase of the main separation column.
Guard columns or cartridges are inserted in the eluate fluid stream, generally between the sample injection valve and the separation column. The guard column or cartridge captures strongly retained dissolved sample components and prevents them from passing through and contaminating the separation column. The guard column or cartridge also captures particulate material. Guard columns and cartridges are extensively used and commercially available.
Despite widespread use, guard columns and cartridges have several drawbacks. Guard columns may degrade the separation efficiency and performance of chromatography columns. Column efficiency or band broadening can be affected by the flow path taken when a guard column or cartridge is used. Typically, the fluid stream travels from a sample injection valve through a tube having a small inside diameter to the guard column or cartridge, which has a relatively large inside diameter, and then back to a connecting tube having a small inside diameter and finally to the separation column which again has a relatively large inside diameter. This change in diameter of the flow path produces a broadening effect which is especially harmful in preparative columns having very large diameters.
Guard columns and cartridges contain chromatographic packing material and sample peaks can be retained slightly by this guard column material. This causes the sample peaks to broaden slightly reducing the efficiency of the separation. More importantly, guard columns and cartridges change the retention time of sample components. The change in retention times is a serious problem, especially for chromatography systems employing computer data acquisition systems. These systems rely on a peak time window to locate particular peaks.
Chromatographic separations reported in chemical literature and references typically publish retention times for peaks of interest. Use of guard columns which affect retention times increases the difficulty of matching separations to published retention times in order to verify and confirm experimental results.
Finally, the effectiveness of a guard column or cartridge is generally determined by observing the chromatographic separation. The stationary phase packing material in a guard column or cartridge is shielded from view by the column hardware. Colored contaminants can only be observed if the column end is opened, a process not recommended by most manufacturers. Opening the column disturbs the packing bed and further degrades chromatographic performance of the column. Although separation parameters such as plate number, pressure and resolution can be measured to determine the performance of a guard column or cartridge, these parameters do not always indicate whether a guard column or cartridge is adequately protecting an analytical column. Fouling of an analytical or preparative column can still take place well before there are noticeable changes in plate number, pressure and resolution.
In addition to conventional bulk powdered stationary phase packing materials traditionally used in liquid chromatography, synthetic membrane chromatography materials are also known. These membranes are generally made from synthetic or natural polymers in the form of web-type membranes, paper sheets, etc. For example, ion exchange resin beads or reversed-phase silica particles can be embedded or enmeshed in a membrane or network of polytetrafluoroethylene. These membranes are suitable for use as chromatographic stationary phase materials or as sorptive materials in filtration equipment. See for example D.F. Hagen et al, Anal. Chim. Acta, 1990, 236:157-164 and T.B. Tennikova et al, Journ. Chrom., 1991, 555:97-107.
Membrane and fabric chromatographic materials are also known in which the surface of the fabric or membrane is chemically modified to provide functional groups suitable for chromatographic separation. Examples include modified cellulose paper having weak ion-exchange groups, cross-linked vinyl polymer sheets containing ion-exchange groups, cellulose membranes having attached ligands for affinity chromatography. Membranes have also been prepared by polymerization of glycidyl methacrylate and ethylene-dimethacrylate to produce membranes having surface epoxide groups. The epoxide groups are then modified to provide a solid phase material suitable for protein separation. See T.B. Tennikova et al, Journ. Liq. Chrom., 1990, 13:63-70.
A need continues to exist for improved methods of protecting liquid chromatography columns. There is a particular need! for methods of protecting liquid chromatography columns which do not substantially alter the retention time of sample peaks, do not contribute to substantial peak broadening and yet provide adequate protection for analytical and preparative columns.